The present invention relates in general to constructions and methods for cassette mutagenesis and in particular to constructions and methods for partial marker cassette mutagenesis of xylose isomerase.
Enzymes have been used extensively in industrial processes for over 100 years since the introduction of the use of trypsin in the processing of animal skins into leather. Presently, enzymes are employed in many processes from the microgram scale of medical diagnostic tests to the billion pound scale of high fructose corn syrup production. It is because of their specificity as well as the mildness of their reaction conditions of temperature and pressure that enzymes are readily used in industrial processes.
Nevertheless, a high degree of specificity limits the usefulness of some enzymes. Reaction condition limitations such as pH, temperature, ionic strength and ionic environment limit the range of many enzymes. In some industrial processes, a sequential use of different enzymes dictates that new conditions be established at each new enzymatic step. These extra manipulations can lead to extra costs, especially at the million pound scale.
Traditionally, one would search for an enzyme having more desirable specificity or a greater reaction condition tolerance by screening for the enzyme in naturally occurring microorganisms. Such a search is time consuming and labor intensive. Ideally, one would like to design a protein de novo or at least modify existing ones by mutagenesis so that their activities would match the process specifications.
There exist a number of techniques for the mutagenesis of nucleic acids. The techniques for mutagenesis of polynucleotides, such as genes, may be divided into techniques for deletion mutagenesis, techniques for insertion mutagenesis and techniques for base substitution mutagenesis. Deletion and insertion mutagenesis are generally employed to locate functional elements or protein coding regions of a genome. Shortle, Ann. Rev. Genet., 15, 265-94 (1981). Base substitution mutagenesis is a preferred method of mutagenizing an identified structural gene.
Base substitution mutagenesis may be performed by random chemical or physical mutation or by site-directed mutagenesis employing oligonucleotides synthesized to partially match a nucleotide sequence within a target nucleotide sequence. Random mutagenesis is preferred in any situation where, because of insufficient understanding of the system being studied, there is no obvious rationale for specific base substitutions. Kadonaga et al., Nucleic Acids Res., 13, 1733-1745 (1985). The strategy for random mutagenesis involves exposing a large number of copies of a target sequence to a mutagen and then screening for the presence of mutants.
A polynucleotide may be prepared for mutagenesis by inserting it into a vector such as a circular, double-stranded DNA structure called a plasmid (Humphreys et al., Molec. Gen. Genet., 145, 101-108 (1976)), or such as a virus (Kadonaga et al., supra), both of which replicate independently of the chromosomal DNA that forms the bulk of the hereditary material in a cell. However, the presence of a large amount of plasmid or viral DNA reduces the efficiency of the mutagen by providing a much larger target, most of which is not intended to be mutagenized, and by increasing the probability of missing desired mutations due to deleterious effects resulting from mutagenesis of the vector.
The size of the target presented to a mutagen may be reduced by exposing only the polynucleotide to be mutagenized to the mutagen. Warburton et al., Nucleic Acids Res., 11, 5837-5854 (1983). Where the polynucleotide to be mutagenized is a trp promoter sequence, fusing the mutagenized polynucleotide in phase to a lac Z .alpha. fragment region in a phage M13 vector permits mutants to be scored based upon the expression of the .beta.-galactosidase gene, which is detected as an abnormal intensity of blue color in M13 plaques in agar containing 5-bromo4-chloroindoxyl-.beta.-D-galactoside. Warburton et al., supra. However, the detection of mutants by a cooperative interaction between the mutagenized polynucleotide and a vector-borne label is of limited applicability and is not directly applicable to the mutagenesis of structural genes.
In fact, a major problem with in vitro mutagenesis in general is the absence of a simple method for the screening of mutants in a wild-type background. Kadonaga et al., supra. Although this problem may be avoided by using a very high level of mutagenesis, multiple base substitutions tend to occur which are less desirable for the unambiguous interpretation of mutant phenotypes than are single or double nucleotide substitutions. Kadonaga at al., supra.